Preparation of LNA Phosphoramidites

Abstract

A highly efficient method for the preparation of LNA (Locked Nucleic Acid) phosphoramidite monomers with 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite and 4,5-dicyanoimidazole has been devised. The quality of the phosphoramidites prepared in this manner is equal to HPLC purified phosphoramidites and can easily be used for oligonucleotide synthesis without further purification. In addition the possibility of using 4,5-dicyanoimidazole in catalytic amounts has been investigated and showed optimum results when 0.7 equivalent was used, and that reducing the amount further leads to undesired phosphitylation of the nucleobase.
Furthermore it is demonstrated that LNA phosphoramidite monomers are exceedingly stable in acetonitrile solution thereby prolonging the effective lifetime of the reagent significantly.

Key words

phosphoramidites - phosphitylations - phosphitylation activators - nucleotide analogues - bicyclic compounds

References

• 2 Obika S. Hari Y. Sugimoto T. Sekiguchi M. Imanishi T. Tetrahedron Lett.  2000,  41:  8923 
• 3 Koshkin A. Singh SK. Nielsen P. Rajwanshi VK. Kumar R. Meldgaard M. Olsen CE. Wengel J. Tetrahedron  1998,  54:  3607 
• 4 Singh SK. Nielsen P. Koshkin A. Wengel J. Chem. Commun.  1998,  455 
• 5 Obika S. Nanbu D. Hari Y. Morio JAK. Doi T. Imanishi T. Tetrahedron Lett.  1998,  39:  5401 
• 6 Wengel J. In Antisense Drug Technology, Principles, Strategies, and Applications   Crooke ST. Marcel Dekker; New York: 2001.  p.339 
• 7 Cook PD. Nucleosides Nucleotides  1999,  18:  1141 
• 8 Oerum H. Jacobsen MH. Koch T. Vuust J. Borre MB. Clin. Chem.  1999,  1898 
• 9 Braasch DA. Corey R. Chem. Biol.  2000,  55:  1 
• 10 Wahlestedt C. Salmi P. Good L. Kela J. Johnsson T. Hokfelt T. Broberger C. Porreca F. Lai J. Ren K. Ossipov M. Koshkin A. Jakobsen N. Skouv J. Oerum H. Jacobsen MH. Wengel J. Proc. Natl. Acad. Sci. U.S.A.  2000,  97:  5633 
• 11 Uhlmann E. Curr. Opin. Drug Discovery Dev.  2000,  3:  203 
• 12 Baker BF. Monia BP. Biochim. Biophys. Acta  1999,  1489:  3 
• 13 Bennett CF. Cowsert LM. Biochim. Biophys. Acta  1999,  1489:  19 
• 14 Salmi P. Kela J. Good F. Wengel J. Wahlestedt C. Society for Neuroscience  1999,  25:  2003 
• 15 Elayadi AN. Corey DR. Curr. Opin. Chem. Biol.  2001,  2:  558 
• 16 Oerum H. Wengel J. Curr. Opin. Chem. Biol.  2001,  3:  239 
• 17 Obika S. Uneda T. Sugimoto T. Nanbu D. Takefumi T. Imanishi T. Bioorg. Med. Chem.  2001,  9:  1001 
• 18 Koshkin AA. Fensholdt J. Pfundheller HM. Lomholt C. J. Org. Chem.  2001,  66:  8504 
• 19 Sinha ND. McManus J. Köster H. Nucleic Acids Res.  1984,  12:  4539 
• 20 Nielsen J. Taagaard M. Marugg JE. van Boom JH. Dahl O. Nucleic Acids Res.  1986,  14:  7391 
• 21 Håkansson AE. Koshkin AA. Sørensen MD. Wengel J. J. Org. Chem.  2000,  65:  5161 
• 22 Singh SK. Kumar R. Wengel J. J. Org. Chem.  1998,  63:  10035 
• 23 Sanghvi YS. Guo Z. Pfundheller HM. Converso A. Org. Process Res. Dev.  2000,  4:  175 
• 25 Krotz AH. Klopchin PG. Walker KL. Srivatsa GS. Cole DL. Ravikumar VT. Tetrahedron Lett.  1997,  38:  3875 
• 26 Vargeese C. Carter J. Yegge J. Krivjansky S. Settle A. Kropp E. Peterson K. Pieken W. Nucleic Acids Res.  2000,  26:  1046 
• 27 Pedersen DS. Rosenbohm C. Synthesis  2001,  2431 
• 28 Brown T. Brown DJS. In Oligonucleotides and Analogues - A Practical Approach   Eckstein F. Oxford University Press; Oxford: 1991.  p.9 
• 29 Nurminen EJ. Mattinen JK. Lönnberg H. J. Chem. Soc., Perkin Trans. 2  1998,  1621 
• 30 Berner S. Mühlegger K. Seliger H. Nucleic Acids Res.  1989,  17:  853 
• 31 Dahl BM. Nielsen J. Dahl O. Nucleic Acids Res.  1987,  15:  1729 
• 32 Barone AD. Tang J.-Y. Caruthers MH. Nucleic Acids Res.  1984,  12:  4051 

1

We have defined LNA as an oligonucleotide containing one or more 2′-O,4′-C-methylene-β-d-ribofuranosyl nucleotide monomer(s). LNA has recently also been named 2′,4′-BNA. [2]

24

1H-Tetrazole is an explosive chemical and has also been reported to be toxic. [23]

33

At no time during our catalytic studies with DCI did we detect any nucleobase phosphitylated products. We have attributed this to the expected higher reactivity of the nucleobase phosphoramidites resulting in their hydrolyses during the aqueous bicarbonate workup prior to HPLC analysis.

34

All phosphitylation reactions were completed in less than 2 h except for the G phosphoramidite 7b synthesis, which took up to 4 h for completion.